This invention relates to techniques in the separation sciences and more particularly to techniques for: (1) injecting samples into a separating apparatus; (2) separating molecular species of the samples with low band spreading; and (3) monitoring the separated molecular species of the samples.
It is known in the separation sciences to automatically inject samples into a separating apparatus, separate the different molecular species from each other and detect the bands after they have been separated. One type of such separating apparatus performs separation by electrophoresis and is known as an electrophoresis apparatus. In this process, the samples are separated in a medium as the molecular species are moved through the medium under the influence of an electrical potential.
One class of electrophoresis apparatus is a capillary electrophoresis apparatus. In a capillary electrophoresis apparatus, the medium is in a capillary tube. This tube is usually made of fused quartz. The electrophoresis medium may be a gel or liquid in capillary electrophoresis.
To sense the separated bands of molecular species, a detector transmits light through the medium phase and senses the species as they move along the medium by differences in absorbance of the light. In one class of electrophoresis apparatus, the sensing of molecular species is accomplished by transmitting light from a light source section through the medium and into a light detecting section. The light detector is positioned in the light detection section where it receives light and converts the light to an electrical signal representing the transmittance of light through a band.
In one type of prior art detector of this class, light is transmitted through one narrow slit and one larger opening in the light path between the source and the light sensor or no narrow slits so as to utilize maximum light flux.
Without the single narrow slit, some of the light flux passes through the medium to the detector, but other parts of the light flux bypass the medium. The flux passes around the medium through the walls of the quartz capillary tube and to the detector. This bypassed light results in a high level of background light which reduces the sensitivity of the detector. A conventional method of reducing background light is to use one narrow slit and collimated light. This arrangement, if applied to capillary electrophoresis, has a disadvantage in that the light level for detection is so low that there is excessive noise in relation to the strength of the electrical signals supplied by the detector. This excessive noise is quantum noise.
Another aspect of the prior art capillary electrophoresis apparatus is that, under some circumstances, a relatively high potential is used to increase the speed of migration of the molecular species through the electrophoresis medium. This is intended to increase the sharpness of the peaks and provide better resolution. The electrophoresis medium in prior capillary electrophoresis apparatus (inside diameter of capillary tube less than 0.01 inch) has a vertical orientation at least at some locations.
This prior art technique has an unobvious disadvantage. It has been discovered that vertical portions of the column reduce the resolution of the bands of molecular species being separated in a liquid electrophoresis medium in an unexpected manner. It is believed that this reduction in resolution occurs because the high electrical potential through the column raises the temperature and causes movement of the medium phase by convection. The convection currents have vertical components of motion at some locations which differ in direction or velocity from the vertical components in other locations. Some of the components of motion are axial with respect to the capillary tube, causing axial mixing and degradation of resolution.
Since the center of the medium is warmest because of its distance from cooling surfaces, the liquified medium in the central portion rises upwardly. The medium is cooled along its sides and moves downwardly as the center moves upwardly. The convection takes place in cells located adjacent to each other along the longitudinal axis of a verticle elongated separating medium. This effect is believed to take place particularly in capillary electrophoresis apparatuses.
Because the convection currents in the medium have a component of motion in the direction of migration of the molecular species in a vertical portion of the medium, the velocities of movement of the molecular species in different portions of a band are changed with respect to each other. This causes band spreading and a reduction of resolution.
The disadvantages caused by the convection currents may be minimized by using a small internal diameter capillary tube to hold the medium because: (1) more heat per unit of length is generated in a larger capillary tube; and (2) there is a longer thermal path to the inside wall of a larger capillary tube than a smaller capillary tube and more space for convection cells to form. On the other hand, small capillaries result in small peak volumes making detection less sensitive.
One type of sample injector injects very small samples by applying a vacuum on the end of the separating apparatus opposite to the source of the sample to draw a small portion of the sample into the separating apparatus.
The prior art sample injectors of this type control the amount of sample by timing the period through which the vacuum is applied. The time is determined by experience. This type of prior art sample injector has a disadvantage of not being as precise as desired.